P465: N-MYRISTOYLATION INHIBITION ABROGATES OXIDATIVE PHOSPHORYLATION TO TARGET ACUTE MYELOID LEUKEMIA STEM CELLS
Authors: Jay Gamma, Erwan Beauchamp, Qiang Liu, Morris Kostiuk, Aishwarya Iyer, Megan Yap, Zoulika Zak, Cassidy Ekstrom, Joseph Brandwein, Jean Wang, John Mackey, Luc Berthiaume
Journal: HemaSphere (2023)

Background: Protein N-myristoylation is the linkage of the fatty acid myristate to the N-terminus of a protein. Catalyzed by two human enzymes – N-myristoyltransferase 1 & 2 (NMT1/2) – this modification alters the stability, localization, complex association, and function of proteins. Up to 600 myristoylated human proteoforms impact a wide variety of processes including signal transduction, cytoskeletal rearrangement, apoptosis and metabolism. We reported the importance of myristoylation of Src-family kinases (SFKs) for B-cell receptor signaling in B-cell lymphomas; treating cells with first-in-class myristoylation inhibitor PCLX-001 rapidly led to cell death. In acute myeloid leukemia (AML), two frequently mutated receptor tyrosine kinases, FLT3 and c-Kit, rely upon SFKs for signal transduction, leading us to explore the therapeutic potential of PCLX-001 in AML. A challenge in treating AML is leukemic stem cells (LSCs); a rare, self-renewing population which frequently survives therapy, expanding to re-establish disease. The heterogeneity of LSCs adds further complexity as therapies result in the enrichment of drug-resistant clones leading to refractory disease. New AML therapies thus require targeting LSCs. Recent reports showed that LSCs have a relatively inflexible metabolism, rely on oxidative phosphorylation (OXPHOS) for survival and that inhibition of OXPHOS led to LSCs killing. Aims: To demonstrate the efficacy of myristoylation inhibitor PCLX-001 in AML, elucidate its mechanism of action, and evaluate its ability to target LSCs. Methods: Efficacy of PCLX-001 against AML cell lines in comparison to PBMCs and lymphocytes from healthy individuals was determined by CellTiterBlue viability assay. SFK proteins, SFK activation, ER stress, and apoptosis induction were examined by western blotting. PCLX-001 efficacy against LSCs was examined using OCI-AML-22 cell model by assaying viability of LSC-enriched fractions. In vivo efficacy was determined using tail vein and intra-femoral murine xenografts of primary patient samples; mice were treated with PCLX-001 and blood or marrow collected for flow cytometric analysis for engrafted cells. Seahorse and Resipher platforms were used to measure oxygen consumption rates (OCR) in AML cell lines. Levels of myristoylated mitochondrial complex I proteins NDUFAF4 and NDUFB7 were measured by western blotting. Levels of total complex I and its in-gel activity were measured after blue native gel electrophoresis of isolated mitochondria. Results: PCLX-001 treatment induced cell death in AML cell lines at concentrations that spared healthy PBMCs and lymphocytes. PCLX-001 treatment resulted in loss of total SFKs and SFK phosphorylation leading to an increase in ER stress (Bip protein) and apoptosis (caspase 3 cleavage). In OCI-AML-22, LSC-enriched population viability was reduced in response to PCLX-001 treatment after 48 hours, and bulk populations after 72 hours. In vivo, PCLX-001 treatment reduced the numbers of AML cells circulating in the blood and marrow. PCLX-001 produced a dose-dependent reduction in OCR in treated AML cells, and a corresponding loss of NDUFAF4 and NDUFB7 proteins, and complex I assembly and activity. Summary/Conclusion: We demonstrate PCLX-001 kills bulk leukemic cells and LSCs in vitro and in vivo by disrupting the function of key myristoylated proteins. While PCLX-001 is currently undergoing clinical evaluation in B-cell lymphoma and solid tumours, these results warrant clinical evaluation in AML with trials scheduled to start spring 2023. Keywords: Acute myeloid leukemia, Cancer, Src kinase, Mitochondria